The overall objective of the proposed research is to elucidate the amino acid sequence of cAMP-dependent protein kinase in porcine skeletal muscle. Two forms of cAMP-dependent protein kinase have been purified to homogeneity, and a large scale procedure for purification of regulatory and catalytic subunits has been developed. Sequencing of the catalytic subunits has already begun. Two-dimensional maps of the tryptic peptides from each catalytic subunit have been characterized and purification of these peptides is in progress. The sequencing will proceed with purification and characterization of the cyanogen bromide peptide fragments from the catalytic subunits. If necessary maleylated tryptic peptides, thermolysin peptides, and chymotrypsin will be isolated to establish overlapping sequences. Diagonal electrophoretic procedures will be used for selectively purifying tryptic peptides containing methionine residues. The sequence of the regulatory subunits also will be elucidated. In conjunction with sequencing, efforts will be made to identify functional residues or regions in the molecule. Differential labeling of surface lysine residues with (14C)-maleic anhydride in the holoenzyme and in the dissociated subunits will be used to identify regions of subunit interaction. Any essential cysteine residue(s) will be identified by titration and correlated to the amino acid sequence. Any disulfide bonding will be characterized by diagonal electrophoresis. 8-Azido-cAMP will be used as an affinity label for identifying regions of the sequence involved in cAMP binding in the regulatory subunit. Limited proteolysis of the regulatory subunit will also be investigated. Any proteolytic fragments will be characterized and attempts will be made to correlate proteolytic fragments to functional domains of the molecule. Efforts are being made to crystallize both the holoenzymes and the dissociated regulatory and catalytic subunits. If suitable crystals are grown, they will be studied by x-ray diffraction in the Crystallography Division of our department.